Dendrimer fluid purification method

ABSTRACT

A dendrimer complex comprising a dendrimer and an antimicrobial agent with the dendrimer complex placeable directly into a fluid to inhibit growth of microbes or rid the fluid of microbes. In a further embodiment of the invention the dendrimer complex is secured to a carrier, which is placed in a body of fluid and allowed to dispense the antimicrobial agent into the fluid. Once the antimicrobial agent is dispensed, the dendrimer complex can be removed and recycled to add functional groups to the dendrimer so that the dendrimer can be reused.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 10/393,903filed Mar. 24, 2003, now U.S. Pat. No. 7,048,864.

FIELD OF THE INVENTION

This invention relates generally to fluid purification such as byridding fluids of microbial activity and, more specifically, to use ofdendrimer complexes to rid fluids of unwanted microbial activity.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

None

REFERENCE TO A MICROFICHE APPENDIX

None

BACKGROUND OF THE INVENTION

The concept of dendrimer technology and particularly dendrimers are wellknown in the art. In general, a dendrimer is a large molecule having asphere-like shape and includes a core with outer branches for holdingfunctional groups.

Dendrimers are unique, highly branched, organic molecules that representa breakthrough in polymer synthesis. Dendrimers are considered to benovel because of their size, shape, and physical or chemical properties.Dendrimers represent an example of synthetic organic molecules havinguniform size and 3-dimensional structures. A promising feature ofdendrimers is that they can be synthesized to have various internalstructure and various surface properties. Because of the multiplereaction sites on the dendrimer molecules they can be combined with anumber of different compounds.

Dendrimers are considered nanotechnology since the size of a dendrimermolecule is typically in the nanometer range. In general, the dendrimersare comprised of three components a) a core material; b) ahighly-uniform branched polymers attached in step-wise layers to thecore and c) a nano-scaled 3-dimensional surface structure, commonlyspherical. While the synthesis of dendrimers is known, it is oftentimesdifficult and time consuming, thus the cost of dendrimers can berelatively expensive costing thousands of dollars per pound. Thetechnology and use of dendrimers remains limited at least partly due tothe high costs of the dendrimers. However, various dendrimer patents andpublications on dendrimers include laundry lists of proposedapplications or uses for dendrimers.

An example of a proposed use of a dendrimer in “wound treatment” isshown in Balogh U.S. Pat. No. 6,224,898. Balogh et al discloses that anantimicrobial agent comprising a metal-containing compound isdistributed on or in a dendrimer polymer for applying to a specific typeof wound, namely, “burn wounds”. Balogh et al states his “dendrimersilver nanocomposite” is applied to a wound by a spray or is applied toa cloth substrate and then applied to a wound. While Balogh et al.conducts tests he concludes that his antimicrobial agent is effective“in vitro” for treatment of wounds.

While the dendrimers generally do not have any antibacterial propertiesthe Cooper et al. U.S. Pat. No. 6,440,405 discloses formation of aquaternary ammonium functionalized dendrimer that does derive itsantibacterial properties from the dendrimer itself. In Publication No.US 2002/022012 Cooper et al. goes on to propose “surface treatments”using a dendrimer biocide-silver nanocomposite as an antimicrobialagent. Cooper et al. prepares his biocide-silver nanocomposite byreacting the dendrimer biocide disclosed in his U.S. Pat. No. 6,440,405with a silver compound. Cooper et al. then incorporates hisbiocide-silver nanocomposite into various surface coatings to providethe coating or surface with antimicrobial activity. Cooper et al. pointsout the use of the dendrimer with biocide-silver nanocomposite as beingeffective against agents such as anthrax. Cooper et al. specificallypoints out the incorporation of his dendrimer silver nanocomposite into“protective coatings or paints, personal products such as cosmetics,industrial products, hospital products, and sanitation of swimming poolsand spas.” Cooper et al. goes on to point out that his dendrimer silvernanocomposite can be “immobilized onto the surface to create efficientantimicrobial surfaces for use as biomaterial, anti-fouling paints, andother similar devices.” Thus, Cooper et al. teaches the use of thebiocide-silver nanocomposite within the structure or some type ofcoating to make antimicrobial surfaces.

The Tomalia et al. U.S. Pat. No. 5,714,166 discloses the use ofdendrimers for “pharmaceutical and agricultural applications” Morespecifically, Tomalia et al discloses dense star polymers can beassociated with a bioactive agent, a diagnostic agent or atherapeutic/diagnostic agent. Tomalia et al. teaches the use of thedense star polymers in pharmaceutical and agricultural applications. Hespecifically points out the transporting of genetic material through acellular member and into a cellular nucleus with a dendrimer polymer.

The Tomalia et al U.S. Pat. No. 4,507,466 discloses the uses ofdendrimers for emulsifier for oil or water emulsions, as wet strengthagents in the manufacture of paper and agents for modifying theviscosity in aqueous formations such as paints.

The Tomalia et al. U.S. Pat. No. 4,694,064 discloses the use ofdendrimer in the production of molecular composites and as crystallinemodifiers for polymeric materials.

In the above references it is evident that the dendrimers or dendrimerbiocides have been combined with various biocidal agents to produceantibacterial products that can be incorporated into articles orcoatings to rid surfaces of bacteria. In contrast, the present inventionhas found that one can incorporate dendrimers with an antimicrobialagent directly into a fluid to control microbial activity in the fluidin order to render the fluid safe for either consumption or recreationaluses. In addition, the invention includes a fluid treatment method thatpermits one to rid or at least inhibit microbial growth in the fluid andwhen the dendrimer antimicrobial agent is spent permits one to removeand recycle the dendrimer for combining with fresh antimicrobial agents.

SUMMARY OF THE INVENTION

A dendrimer complex comprising a large scale molecule having anantimicrobial agent as a functional group with the dendrimer complexplaceable directly into a fluid to rid the fluid of microbes. In afurther embodiment of the invention the dendrimer complex is secured toa carrier, which is placed in a fluid and allowed to release theantimicrobial agent carried by the dendrimer directly into the fluid.Once the antimicrobial agent is spent, the dendrimer complex can beremoved and recycled to add fresh functional groups to the dendrimer sothat the dendrimer can be reused.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A schematically represents two molecules with sites for functionalgroups;

FIG. 1 schematically represents the two molecules of FIG. 1 withfunctional groups secured to the outer branches;

FIG. 2 shows a container containing a fluid with a dendrimer withfunctional groups schematically illustrated in the bottom of thecontainer;

FIG. 3 is a dispenser containing a dendrimer with a antimicrobial agentfor placing in a fluid;

FIG. 4 shows a carrier such as a grid or fabric with dendrimers withfunctional groups secured to the carrier; and

FIG. 5 shows a bead like object having a plurality of dendrimers withfunctional groups secured to the bead like object.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Dendrimers are known in the art and generally comprise large sphere likemolecules that have a core, a shell of internal cells and a shell ofexterior cells with sites for functional groups. One of the functionalgroups used with dendrimers includes biocides, other include specificmaterials. An example of dendrimer with a silver complex is shown in theBalogh U.S. Pat. No. 6,224,898, which is incorporated herein byreference.

The present invention utilizes the dendrimer as a carrier for anantimicrobial agent to permit the dendrimer with the antimicrobial agentto be placed directly into a fluid environment or in a fluid, whichrequires purification for a variety of purposes including being consumedor used for recreational purposes. As used herein the term dendrimerdescribes a highly branched molecule having a number of sites forattaching functional groups thereto. A dendrimer complex describes adendrimer with a functional group attached to the dendrimer. Forexample, a dendrimer silver complex refers to a dendrimer with silversecured to the dendrimer. Similarly, a dendrimer metal ion yieldingcomplex refers to a dendrimer having a metal secured to the dendrimerwith the metal releasing metal ions when the dendrimer metal ionyielding material comes into contact with a fluid, such as water. Asused herein the term antimicrobial agent references to a material thatkills or inhibits the growth of microbes including bacteria and otherorganisms.

FIG. 1 schematically represents a first large scale spherical molecule11 located proximate a second large scale spherical molecule 12. Locatedon spherical shaped molecule 11 are a set of sites 11 a where functionalgroups can be attached. Similarly, reference numeral 12 designates asecond spherical shaped molecule having a set of sites 12 a forsecurement of a functional group thereto. FIG. 1 represents dendrimersin a generally pre active state.

A reference to FIG. 1A illustrates the first spherical molecule 11 witha set of functional groups 11 b secured to the sites 11 a on molecule11. Similarly molecule 12 contains a set of functional groups 12 bsecured to sites 12 a on the molecule 12. In the present invention, anantimicrobial agent, which comprises the functional group, is secured tothe dendrimer for purposes of controlling or killing microbes in afluid. Examples, of specific types of materials useful in a fluid tokill microbes such as bacteria are metal ion yielding materials. One ofthe known metal ion yielding materials is silver chloride another issilver nitrate. Other metal ion yielding materials for use in waterpurification the for killing microbes can be a metal ion yieldingmaterial selected from the group consisting of zinc sulfate, zinccarbonate, zinc chloride, copper chloride, copper carbonate, coppersulfate, silver chloride, stannous chloride and stannic chloride. FIG.1A schematically represents the dendrimer complex for killing microbesby release of the functional groups from the dendrimer molecule.

A part of the present invention is a method of disrupting microbeproliferation comprising securing a ion yielding material to a dendrimerand then bringing the dendrimer containing the ion yielding material inion communication with a source of microbes, for example by placing thedendrimer with the ion yielding material into a body of fluid 15 asshown in FIG. 2 to allow the ions to come into the presence of themicrobes. The body of fluid 15 can be recreational water such as foundin a spa, hot tubs and pools or it can be fluid intended for consumptionsuch as drinking water or it can be other types of fluids where thefluid requires purification such as by ridding or curtailing themicrobial activity in the fluid. Thus, in one embodiment the presentinvention comprises a method of disrupting microbe proliferation bysecuring an antimicrobial agent to a dendrimer and immersing thedendrimer containing the antimicrobial agent in a fluid containingmicrobes, such as found in a body of water, to control microbialproliferation and thus render the water drinkable or suitable forrecreational purposes.

In another method of fluid treatment one secures an antimicrobial agentto a dendrimer and places the dendrimer containing the antimicrobialagent into a fluid, which containing microbial activity, forsufficiently duration to rid the fluid of microbes. The fluid is notlimited to water and could be fluids such as drinkable juices or thelike. One then maintains the dendrimer containing the antimicrobialagent in the fluid for at least a time sufficient to control themicrobial activity. The dendrimer containing the antimicrobial agent canthen be removed and recycled by replacing the spent antimicrobial agent.

A further feature of the invention is that although the dendrimercontaining the antimicrobial agent can be removed from the fluid one canalso leave effective amounts of dendrimers containing antimicrobialagents in the fluids sufficient to kill the microbes in the fluidswithout having the antimicrobial agent or the dendrimer exceeding safelimits for human consumption or recreational use.

Another method of antiseptic treatment comprises forming a group ofdendrimers and securing a antimicrobial agent to each of the group ofdendrimers and bringing the group of dendrimers with the antimicrobialagent secured thereto into a source of microbial activity in a fluid sothat the fluid must flow around the dendrimer containing the functionalgroup. For example, one may want to kill microbes in body fluidsincluding blood by incorporation the dendrimer with the antimicrobialagent into the fluid by running the blood through a screen having adendrimer with an antimicrobial agent secured to the screen to therebyprovide for on-the-go killing of microbes in the body fluid as the bloodflows through the screen.

In another embodiment, the present invention comprises the method ofmicrobial treatment in fluids comprising securing an antimicrobial agentsuch as a biocidal active agent to a dendrimer and applying thedendrimer with the biocidal active agent to a carrier, bringing thecarrier with the biocidal active agent thereon into the presence of amicrobial active site, such as in a fluid, and maintaining the carrierwith the biocidal active agent in the presence of the microbial activesite for sufficient time to at least inhibit microbial growth.

To determine the antimicrobial potential of various dendrimer complexesa dendrimer-silver complex was prepared using commercially availablesilver acetate with a dendrimer purchased from Dendritech, Inc. ofMidland, Mich. After formation of the dendrimer-silver complex testswere performed to evaluate the antimicrobial activity usingmicroorganisms of both Pseudomonas aerligenosa and E. coli. The testprocedure is as follows:

EXAMPLE ONE

1. Prepare an inoculum of the test organism by streaking the entiresurface of agar plates by the standard method for colony isolation, withE. coli (Nutrient Agar) and Pseudomonas (Pseudomonas IdentificationAgar). Incubate the streaked agar plates at 37° C. for approximately 18hours.

2. Harvest all the growth on the test organism plates by flooding theplates with 9.0 ml of sterile distilled water per plate. Wash theharvested biomass by centrifugation for 1 hour at 3000 rpm, discardingthe supernatant fluid, and resuspending the biomass in 9.0 ml of steriledistilled water. Resuspend harvested cell by vortex agitation for 1minute.

3. Dilute 0.1 ml of the dendrimer-silver complex into 100 ml of 0.1%saline and sterile using a 0.45 micrometer filter.

4. Dispense 9.8 ml of the diluted complex to sterile test tubes andinoculate with 0.2 ml of the washed test microorganism as describedabove and mix.

5. Incubate for 10 minutes and perform serial dilution's and dispense0.2 ml of dilution to agar plates for microbial enumeration by thespread plate method. E. coli was cultivated on Nutrient Agar (NA) andPseudomonas was cultivated on Pseudomonas Identification Agar (PIA)

6. Incubate agar plates overnight at 37° C. and perform enumeration onplates having between 30 to 300 colonies.

7. Determine antimicrobial activity by taking the log difference betweenan untreated control and a plate of the same dilution treated with thetest nanocomposite.

Test Results

Based on enumeration by the spread plate method, the untreatedPseudomonas control plate contained 1.94×10⁷ colony forming unit (cfu),equivalent to a log₁₀ value of 7.29. The Pseudomonas plate treated withthe dendrimer-silver complex contained 1.0×10⁵ cfu, equivalent to alog₁₀ value of 5. The log₁₀ different after 10 minutes of treatment is2.3 or more than 99% reduction in cell numbers between the control andthe treated plate.

The untreated E. coli had 1.3×106 cfu and the E. coli treated with henanocomposite had less than 1×10⁵ for a log 10 reduction of at least 1.1which is equivalent to a 90% reduction in cfu after 10 minutes.

In contrast, testing with silver ions alone requires at least one hourto obtain a 1 log₁₀ reduction in cfu of the test organisms. Based on thetest results it was concluded that silver in the form ofdendrimer-silver complexes, when placed in a fluid environment, issubstantially more active as an antimicrobial substance than silver ionsalone, even though it is well known that the silver ions posses strongantimicrobial activity.

EXAMPLE TWO

A second sample of a dendrimer silver complex was tested against variousconcentrations of silver nitrate against both E. coli and Pseudomonasaerugentoas. The experiment was performed as follows:

1. Prepare an inoculum of the test organisms by streaking the entiresurface of agar plates, by the standard method for colony isolation,with ether E. coli or pseudomonas aerugentoas. The plates were incubatedat 370° C. for approximately 18 hours.

2. Harvest all the growth on the test organism plates by flooding theplats with 9.0 ml of sterile distilled water per plate. Wash theharvested biomass by centrifugation for 1 hour at 3000 rpm, discardingthe supernatant fluid, and resuspending in 9.0 ml of sterile distilledwater. Resuspend harvested cell by vortex agitation for 1 minute.

3. Inoculate test plates with 0.2 ml with the test organism prepare asdescribed above.

4. Place sterile 6-millimeter blank paper Discs on test plates andsaturate with 10 microliters of the test substances.

5. Incubate the plates for 24 hours at 370c and report results as thediameter of the zone of inhibition around the discs treated with thetest substances. The results are summarized below:

TEST RESULTS Pseudomonas (diameter of E. Coli Test Substance inhibitoryzone) (diameter of inhibitory zone) 10.0% Silver Nitrate 11 6  7.5%Silver Nitrate 7 6  5.0% Silver Nitrate 6 6  2.5% Silver Nitrate 6 6 1.0% Silver Nitrate 9 6  0.5% Silver Nitrate 12 6 Complex I 11 10Complex I dialyzed 17 8 Complex II 13 9 Complex III 18 10

The tests results show the diameter of the inhibitory zone inmillimeters where the microbes were effectively destroyed. The testsreveled that the dendrimer silver complex has an enhanced antimicrobialactivity when compared to silver nitrate as evidenced by the largerdiameter inhibitor zones. The exact mechanism is not fully understoodbut reveals that the dendrimer silver complex not only has greatereffectiveness in killing microbes in fluids than well known metal ionyielding compounds but also acts more quickly than other metal ionyielding compounds that yield the same ion.

In the present invention various methods of deliver of the dendrimercomplex containing the antimicrobial agent to the fluids are used. Inone method the dendrimer complex containing the antimicrobial agent 31is adhered to a small bead or fluid insoluble particle 30, such as shownin FIG. 5, or other particle carrier that has sufficient size to beretained within a dispenser as shown in FIG. 3. Dispenser 20 has a setof openings 21 which are smaller than the dendrimer containing material22 within dispenser 20 to thereby retain the dendrimer containingmaterial 22 therein. By confining the dendrimer complex 22 within thedispenser 20 the dendrimer complex 22 can be recycled and reused. Thedendrimer complex 22 can be bonded to various material, such asillustrated in FIG. 5, to maintain the dendrimer in a condition wherethey can be retained and recovered for recycling. By adhering thedendrimer to a fluid insoluble carrier one can maintain the dendrimer inthe fluid for purposes of fluid purification and then remove thedendrimer for recycling of the dendrimer.

FIG. 4 shows another method, wherein the dendrimer is adhered to alarger structure such as some type of flow through member 25 that comesinto contact with the fluid. For example, the dendrimer silver complex26 can be placed on a flow through carrier such as a filter, a cloth, amembrane or other porous material or non porous material arranged topermit fluid to flow therethrough, such as illustrated in FIG. 4. A flowthrough carrier allows the dendrimer complex to come into direct contactwith the fluid to be treated by flowing the fluid through the carrier.Although flowing the fluid through the carrier one can also immerse thecarrier in the fluid. For example, placement of a carrier such asmembrane or a fabric directly into a body of fluid such as water permitsthe dendrimer to yield the antimicrobial agent to attack the microbes inthe fluid as the fluid flows through the carrier.

In still another method the dendrimer antimicrobial complex is placeddirectly in the fluid to be treated without adhering the dendrimerantimicrobial complex to a carrier. The dendrimer antimicrobial complexis useful in direct treatment all types of fluids including body fluids,recreational fluids and drinking fluids. As the dendrimers lacks anyknown appreciable toxicity the incorporation and the presence of thedendrimers in fluids, even those which are ingested by humans, ispossible. In addition, the amount of measurable metal ions present inthe fluids that is sufficient to control microbes can actually be lessthan the amount of measurable ions present with a conventionalnon-dendrimer compounds that also yield metal ions. The greater efficacyof the metal ions deliverable from a dendrimer as opposed to anon-dendrimer enables the use of metal ions for antimicrobial purposesthat might be effective in killing microbes but could not be usedbecause it requires antimicrobial levels in the fluid that would exceedsafe human levels.

Thus the present invention includes a method of disrupting microbeproliferation securing a ion yielding material, such as a metal, to adendrimer, bringing the dendrimer containing an ion yielding material inion communication with a source of microbes in a fluid to controlmicrobial proliferation therein.

The present invention also comprises a method of disrupting microbeproliferation securing an antimicrobial agent to a dendrimer, bringingthe dendrimer containing the antimicrobial agent into proximity of afluid containing microbes and maintain the dendrimer containing theantimicrobial agent in the fluid to control microbial proliferation inthe fluid.

The invention includes a method of killing bacteria in a body of waterapplying a dendrimer containing a water purification material such as abactericide to a carrier, placing the carrier with the dendrimer byimmersing the carrier with the dendrimer containing the waterpurification material in a body of water and allowing water to contactthe dendrimer containing the water purification material such as abactericide to release the bactericide therefrom to kill bacteria in thebody of water. In this method the dendrimer can be removed from the bodyof water after release of the water purification material and a freshwater purification material is releasable secured to the dendrimerfollowed by placing the dendrimer with the fresh water purificationmaterial back into the body of water to continue the water purificationthereof. While a water purification material is attached to thedendrimer includes antimicrobial agents such as bactericides other waterpurification materials such as clarifiers can also be secured to thedendrimer to enable one to deliver the water purification materials tothe fluids. If desired at least two water purification materials such astwo different antimicrobial agents can be secured to the dendrimer toprovide a wider range of effectiveness.

Thus the method of applying dendrimer containing a water purificationmaterial can be placed in recreational waters such as found in a spa, apool or a hot tub. A use of the dendrimer with antimicrobial propertiesin drinkable fluids such as fruit juices functions to rid the juices ofharmful microbes.

A further feature of the dendrimer complex, for example the dendrimersilver complex is even though heavy metals such as silver are consideredtoxic in excess of 100 ppb one can use the dendrimer silver complex tokill microbes in the water yet not have the level of heavy metalconsiderably below the considered toxic levels simply because of theantimicrobial efficiency of the dendrimer silver complex is moreeffective at lower concentrations.

1. A method of disrupting microbe proliferation comprising; securing aion yielding material to a dendrimer complex; placing the dendrimercomplex containing the ion yielding material directly into a fluid to betreated without adhering the dendrimer complex to a carrier, to bringthe dendrimer complex in ion communication with a source of microbes ina fluid to control microbe proliferation.
 2. A method of disruptingmicrobe proliferation comprising: securing an antimicrobial agent to adendrimer to form a dendrimer complex; placing the dendrimer complexcontaining the antimicrobial agent on to a flow through carrier andimmersing the flow through carrier into a fluid containing microbes; andmaintaining the dendrimer complex containing the antimicrobial agentimmersed in the fluid to control microbial proliferation in the fluid.3. A method of killing bacteria in a body of water comprising: applyinga dendrimer containing a bactericide to a flow through carrier; placingthe carrier with the dendrimer in direct contact with a body of water;and allowing the body of water to flow through the dendrimer containinga bactericide carrier to release the bactericide therefrom to kill abacterium in the body of water.